Biology subjects

Huynh, K. W.

Publications and source records attributed to Huynh, K. W..

5 recordsLinked to original sources

bioRxiv2026

Insight into the scaffolding function of USP18 from a high resolution cryo-EM structure of STAT2-USP18-ISG15 ternary complex

USP18 is a primary negative regulator of the type I interferon (IFN-I) signaling which regulates hundreds of IFN-stimulated genes for viral protection and anti-cancer immunity. USP18 plays dual roles in the IFN-I signaling: 1) deubiquitinase enzymatic function which cleaves ISG15 from its substrates and 2) scaffolding function through forming a complex with STAT2 to suppress IFN-I signaling. Targeting the scaffolding function of USP18, instead of its enzyme activity, is crucial for reducing cancer cell fitness and boosting anti-tumor immunity. However, the molecular basis of USP18s scaffolding function remains unclear due to the lack of structural information. Here, using a fusion tag strategy, we captured the transient USP18-STAT2 complex and determined a ternary complex structure of STAT2-USP18-ISG15 at 3.05 [A] resolution by cryogenic electron microscopy (cryo-EM) that delineated detailed USP18-STAT2 interactions. Remarkably, the ternary complex impairs USP18s enzymatic function by STAT2-mediated disruption of its catalytic triad. Structural analysis and mutagenesis identify specific USP18 point mutations, facilitating further investigation into the role of USP18 in IFN-I signaling. Taken together, our findings suggest that USP18s scaffolding function could present an untapped opportunity for cancer therapy.

molecular biology↗

bioRxiv2023

Guanine-containing ssDNA and RNA induce dimeric and tetrameric SAMHD1 in cryo-EM and binding studies

The dNTPase activity of tetrameric SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) plays a critical role in cellular dNTP regulation. SAMHD1 also associates with stalled DNA replication forks, DNA repair foci, ssRNA, and telomeres. The above functions require nucleic acid binding by SAMHD1, which may be modulated by its oligomeric state. Here we establish that the guanine-specific A1 activator site of each SAMHD1 monomer is used to target the enzyme to guanine nucleotides within single-stranded (ss) DNA and RNA. Remarkably, nucleic acid strands containing a single guanine base induce dimeric SAMHD1, while two or more guanines with [~]20 nucleotide spacing induce a tetrameric form. A cryo-EM structure of ssRNA-bound tetrameric SAMHD1 shows how ssRNA strands bridge two SAMHD1 dimers and stabilize the structure. This ssRNA-bound tetramer is inactive with respect to dNTPase and RNase activity.

biochemistry↗

bioRxiv2022

Phosphorylation of SAMHD1 Thr592 increases C-terminal domain dynamics, tetramer dissociation, and ssDNA binding kinetics

SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) is driven into its activated tetramer form by binding of GTP activator and dNTP activators/substrates. In addition, the inactive monomeric and dimeric forms of the enzyme bind to single-stranded (ss) nucleic acids. During DNA replication SAMHD1 can be phosphorylated by CDK1 and CDK2 at its C-terminal threonine 592 (pSAMHD1), enabling the enzyme to localize to stalled replication forks (RFs) and promote their restart. Since localization of a potent dNTPase at stalled RFs is not harmonious with DNA replication, we used a series of kinetic and thermodynamic measurements to explore a hypothesis where the combined effects of T592 phosphorylation and ssDNA binding serves as a dual switch to turn-off SAMHD1 dNTPase activity. We report that phosphorylation has only a small effect on the dNTPase activity and ssDNA binding affinity of SAMHD1. However, perturbation of the native T592 by phosphorylation decreased the thermal stability of tetrameric SAMHD1 and accelerated tetramer dissociation in the absence and presence of ssDNA (~15-fold). In addition, we found that ssDNA binds competitively with GTP to the A1 site. A full-length SAMHD1 cryo-EM structure revealed substantial baseline dynamics in the C-terminal domain (which contains T592) which may be modulated by phosphorylation. We propose that T592 phosphorylation increases tetramer dynamics and allows invasion of ssDNA into the A1 site and the previously characterized DNA binding surface at the dimer-dimer interface. These features are consistent with rapid and regiospecific inactivation of pSAMHD1 dNTPase at RFs or other sites of free ssDNA in cells.

biochemistry↗

bioRxiv2019

Molecular mechanism of TRPV2 channel modulation by cannabidiol

Transient receptor potential vanilloid 2 (TRPV2) plays a critical role in neuronal development, cardiac function, immunity, and cancer. Cannabidiol (CBD), the non-psychotropic therapeutically active ingredient of Cannabis sativa, is a potent activator of TRPV2 and also modulates other transient receptor potential (TRP) channels. Here, we determined structures of the full-length TRPV2 channel in a CBD-bound state in detergent and in PI(4,5)P2 enriched nanodiscs by cryo-electron microscopy. CBD interacts with TRPV2 through a hydrophobic pocket located between S5 and S6 helices of adjacent subunits, which differs from known ligand and lipid binding sites in other TRP channels. Comparison between apo- and two CBD-bound TRPV2 structures reveals that the S4-S5 linker plays a critical role in channel gating upon CBD binding. The TRPV2 \"vanilloid\" pocket, which is critical for ligand-dependent gating in other TRPV channels, stays unoccupied by annular lipids, PI(4,5)P2, or CBD. Together these results provide a foundation to further understand TRPV channel gating properties and their divergent physiological functions and to accelerate structure-based drug design.

biophysics↗

bioRxiv2018

Structural insights on TRPV5 gating by endogenous modulators

TRPV5 is a transient receptor potential channel involved in calcium reabsorption. Here we investigate the interaction of two endogenous modulators with TRPV5. Both phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and calmodulin (CaM) have been shown to directly bind to TRPV5 and activate or inactivate the channel, respectively. Using cryo-electron microscopy (cryo-EM), we determined TRPV5 structures in the presence of dioctanoyl PI(4,5)P2 and CaM. The PI(4,5)P2 structure revealed a novel binding site between the N-linker, S4-S5 linker and S6 helix of TRPV5. These interactions with PI(4,5)P2 induce conformational rearrangements in the lower gate, opening the channel. The CaM structure revealed two TRPV5 C-terminal peptides anchoring a single CaM molecule and that calcium inhibition is mediated through a cation-{pi} interaction between Lys116 on the C-lobe of calcium-activated CaM and Trp583 at the intracellular gate of TRPV5. Overall, this investigation provides insight into the endogenous modulation of TRPV5, which has the potential to guide drug discovery.

biophysics↗